Use of Cationic Steroid Antibiotics to Fight Drug Resistance in Gram-Positive and Gram-Negative Bacteria

Thomas Orsak and Dr. Paul Savage, Chemistry and Biochemistry

Drug resistance has become an increasing problem for clinical anti-bacterial needs and a major human health threat. Drug resistance emerges through structural or biochemical pathway changes that antibacterial drugs specifically target. Bacteria have the ability to rapidly mutate and gain resistance to antibacterial drugs because of their ability to reproduce rapidly. However some parts of bacterial cells need to stay consistent. The need for consistency for these parts arises from their complexity and their essential biological activities. One of these parts is the bacterial membrane.

The Savage group has designed antibiotics that targeted bacterial membranes. Bacterial membrane targets are promising in the fight against drug resistance because of the difficulty the bacteria has to mutate membrane elements without compromising the function of the membrane. The bacterial membrane is as a very complex and specifically permeable barrier. Changes required to become resistant to membrane targeting drugs would be just as complex.

The Savage group has proposed that cationic, facially amphiphillic molecules may act as antibiotic against both gram-positive and double membraned gram-negative bacteria. We have taken a combinatorial chemistry approach and developed large libraries of these compounds. The compounds made by our group are steroid derivatives, called cationic steroid antibiotics (CSAs). These CSAs have shown to cause rapid cell death, be selective for bacterial cells over healthy mammalian cells, have excellent activity against both gram-positive and the harder to kill gram-negative bacteria, and are extremely difficult for bacteria to gain resistance toward. CSAs are also relatively simple to synthesis and manipulate. The ability to manipulate CSAs allows us to easily find what characters of the CSAs create safer and more effective antibacterial drugs. The Savage group has made great contributions in finding desired characteristics for CSAs. Cationic compounds have shown in our testing to be more prokaryotic selective (selective for bacteria over mammals). The Savage group has found that the selectivity and activities of CSAs can be enhanced in two ways. Steroid derivatives with polyamines attached to the C-24 of the steroid backbone have been shown to have more prokaryotic selectivity. The Savage group has also found that longer hydrophobic chain groups attached to C-24 have better activity against gram-negative bacteria than shorter chain groups.

We have tested large libraries of cationic steroid antibiotics to find structural properties that lead to better selectivity between bacterial and mammalian cells and more activity against bacteria. We used a micro-well screening technique with spectrophotometer result determination to rapidly test our large libraries of compounds against both gram-negative using Escherichia coli ATCC 25922 and gram-positive bacteria using Staphylococcus aureus ATCC 25923. Promising micro-well screened compounds were then tested by running larger scale minimum inhibitory concentrations (MICs). We screened over 216 compounds with 1, 2, and 3 amino acid length poly peptide side chains compromising of 6 differing amino acids to determine which were most favorable. F, M, V, W, H, and K amino acids were used for our screenings to produce over 216 compounds out of a possible 8000 if using all 20 common amino acids. Micro-well screening yielded results in dilution ratios of 1/2, 1/4, 1/16, 1/64, and 1/256. MIC retesting of these ratios found 1/16 equaled MICs of over 100 g/mL, 1/64 equaled MIC approximately 40 g/mL, and 1/256 equaled around 10 g/mL and below. Five tri-peptide compounds were found to have activities at 1/256 dilution against Staphylococcus aureus ATCC 25923. These compounds had peptide sequences of FFK, MMK, MWK, MHK, and KHK. Escherichia coli ATCC 25922 testing yielded three compounds with 1/256 activity. These compounds were FFK, MMK, and WKK. Also a trend was shown that K at the α amino acid position on the side chains yielded most of our compounds with 1/64 activity.

The work being done in Dr. Savage’s lab is finding great success. The desired traits found in CSAs have led to testing against additional drug resistant bacteria in synergism with currently available antibiotics. We have also begun testing against cancer cell lines. Both of these initial results are very promising. In addition, resistance studies of these compounds that we are currently testing have found that bacteria are slow to become resistant and lose their resistance within hours, two very desirable traits in the combat against bacteria.

Our lab has shown that CSAs can be easily produced and rapidly screened. We have also shown that just in our library of 216 CSAs several compounds show desired activities against both gram-positive and gram-negative bacteria. The possibilities that these compounds present for human health application is very broad. The low cost of production and storage may help greatly reduce the cost of medical treatments and increase the availability of effective antibacterial treatments. Further testing of an increased library of CSAs and against an increased variety of prokaryotic and eukaryotic cells can result in a more improved understanding of the CSAs capabilities.